Skip to main content
SpringerLink
Log in

Real-time PCR as a diagnostic tool for evaluating the resistance of Chinese cabbage cultivars to Verticillium wilt

  • Published:
European Journal of Plant Pathology Aims and scope Submit manuscript

Abstract

Verticillium wilt disease has become increasingly serious in many regions in China over the past few years. In this study, the widespread pathogen was identified as Verticillium longisporum (V. longisporum) by mt-SSU rDNA and cytochrome b sequence analysis. By using the primer pair HW1-F/HW1-R, which was designed based on the V. longisporum-specific rDNA-ITS sequence, a real-time PCR assay was developed for rapid and specific detection of the pathogen. The real-time PCR assay enabled sensitive quantification of V. longisporum in artificial and field-infected seedlings. This study provides a method for large-scale screening of Verticillium wilt-resistant resources for Chinese cabbage breeding.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Agrios, G. N. (2005). Plant pathology, 5th Edition

  • Ashworth, L. J., Huisman, O. C., Harper, D. M., & Stromber, L. K. (1974). Free and bound microsclerotia of Verticillium albo-atrum in soils. Phytopathology, 64, 563–564.

    Article  Google Scholar 

  • Atallah, Z. K., Bae, J., & Jansky, S. H. (2007). Multiplex real-time quantitative PCR to detect and quantify Verticillium dahliae colonization in potato lines that differ in response to Verticillium wilt. Phytopathology, 97, 865–872.

    Article  CAS  PubMed  Google Scholar 

  • Bhat, R. G., & Subbarao, K. V. (1999). Host range specificity in Verticillium dahliae. Phytopathology, 89, 1218–1225.

    Article  CAS  PubMed  Google Scholar 

  • Butterfield, E. J., & Devay, J. E. (1977). Reassessment of soil assays for Verticillium dahliae. Phytopathology, 67, 1073–1078.

    Article  Google Scholar 

  • Collins, A., Okoli, C. A. N., Morton, A., Parry, D., Edwards, S. G., & Barbara, D. J. (2003). Isolates of Verticillium dahlia pathogenic to crucifers are of at least three distinct molecular types. Phytopathology, 93, 364–376.

    Article  PubMed  Google Scholar 

  • Davis, J. R., Pavek, J. J., & Corsini, D. L. (1983). A sensitive method for quantifying Verticillium dahliae colonization in plant tissue and evaluating resistance among potato genotypes. Phytopathology, 73, 1009–1014.

    Article  Google Scholar 

  • Devay, J. E., Forreste, L. L., Garber, R. H., & Butterfield, E. J. (1974). Characteristics and concentration of propagules of Verticillium dahliae in air-dried field soils in relation to prevalence of Verticillium wilt in cotton. Phytopathology, 64, 22–29.

    Article  Google Scholar 

  • Duniway, J. M. (2002). Status of chemical alternatives to methyl bromide for pre-plant fumigation of soil. Phytopathology, 92, 1337–1343.

    Article  CAS  PubMed  Google Scholar 

  • Duressa, D., Rauscher, G., Koike, S. T., Mou, B., Hayes, R. J., Maruthachalam, K., ... & Klosterman, S. J. (2012). A real-time PCR assay for detection and quantification of Verticillium dahliae in spinach seed. Phytopathology, 102(4), 443–451.

  • Fahleson, J., Hu, Q., & Dixelius, C. (2004). Phylogenetic analysis of Verticillium species based on nuclear and mitochondrial sequences. Archives of Microbiology, 181(6), 435–442.

    Article  CAS  PubMed  Google Scholar 

  • Glynn, N. C., & Edwards, S. G. (2010). Evaluation of PCR assays for quantifying seed-borne infection by Fusarium and Microdochium seedling blight pathogens. Journal of Applied Microbiology, 108, 81–87.

    Article  CAS  PubMed  Google Scholar 

  • Han, R. J., Gen, L. H., Wang, W. H., Yu, S. C., Zhu, Y. L., & Zhang, F. L. (2012). Pathogen identification causing Verticillium wilt on Chinese cabbage in Beijing area. Acta Horticulturae Sinica, 39(3), 477–484.

    CAS  Google Scholar 

  • Ikeda, K., Banno, S., Watanabe, K., Fujinaga, M., Ogiso, H., Sakai, H., Tanaka, H., Miki, H., Shibata, S., Shiraishi, S., & Fujimura, M. (2012). Association of Verticillium dahliae and Verticillium longisporum with Chinese cabbage yellows and their distribution in the main production areas of Japan. Journal of General Plant Pathology, 78, 331–337.

    Article  Google Scholar 

  • Inderbitzin, P., Bostock, R. M., Davis, R. M., Usami, T., Platt, H. W., & Subbarao, K. V. (2011). Phylogenetics and taxonomy of the fungal vascular wilt pathogen Verticillium, with the descriptions of five new species. PloS One, 6(12), e28341.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Johansson, A., Goud, J. K. C., & Dixelius, C. (2006). Plant host range of Verticillium longisporum and microsclerotia density in Swedish soils. European Journal of Plant Pathology, 114, 139–149.

    Article  Google Scholar 

  • Justesen, A., Hansen, H., & Pinnschmidt, H. (2008). Quantification of Pyrenophora graminea in barley seed using real-time PCR. European Journal of Plant Pathology, 122, 253–263.

    Article  CAS  Google Scholar 

  • Karajeh, M. R., & Masoud, S. A. (2006). Molecular detection of Verticillium dahliae Kleb. in asymptomatic olive trees. Journal of Phytopathology, 154, 496–499.

    Article  CAS  Google Scholar 

  • Karapapa, V. K., Bainbridge, B. W., & Heale, J. B. (1997). Morphological and molecular characterization of Verticillium longisporum comb. nov., pathogenic to oilseed rape. Mycological Research, 101, 1281–1294.

    Article  Google Scholar 

  • Klosterman, S. J., Atallah, Z. K., Vallad, G. E., & Subbarao, K. V. (2009). Diversity, pathogenicity, and management of Verticillium species. Annual Review of Phytopathology, 47, 39–62.

    Article  CAS  PubMed  Google Scholar 

  • Li, K. N., Rouse, D. I., & German, T. L. (1994). PCR primers that allow intergeneric differentiation of ascomycetes and their application to Verticillium spp. Applied and Environmental Microbiology, 60, 4324–4331.

    PubMed Central  CAS  PubMed  Google Scholar 

  • Lievens, B., Brouwer, M., Vanachter, A. C. R., Cammue, B. P. A., & Thomma, B. P. H. J. (2006). Real-time PCR for detection and quantification of fungal and oomycete tomato pathogens in plant and soil samples. Plant Science, 171, 155–165.

    Article  CAS  Google Scholar 

  • Maurer, K. A., Radisek, S., Berg, G., & Seefelder, S. (2013). Real-time PCR assay to detect Verticillium albo-atrum and V. dahliae in hops: development and comparison with a standard PCR method. Journal of Plant Diseases and Protection (JPDP), 120(3), 105–114.

    CAS  Google Scholar 

  • Pantou, M. P., Kouvelis, V. N., & Typas, M. A. (2006). The complete mitochondrial genome of the vascular wilt fungus Verticillium dahliae: a novel gene order for Verticillium and a diagnostic tool for species identification. Current Genetics, 50(2), 125–136.

    Article  CAS  PubMed  Google Scholar 

  • Pegg, G. F. (1974). Verticillium diseases. Review of Plant Pathology, 53, 157–182.

    Google Scholar 

  • Pegg, G. F., & Brady, B. L. (2002). Verticillium wilts. New York: CABI Publishing. 432 pp.

    Book  Google Scholar 

  • Qi, J. N., Yu, S. C., Shen, X. Q., Zhao, X. Y., Yu, Y. J., Zhang, D. S., & Zhang, F. L. (2010). Reference gene selection for real-time quantitative polymerase chain reaction of mRNA transcript levels in Chinese cabbage (Brassica rapa L. ssp. pekinensis). Plant Molecular Biology Reporter, 28, 597–604.

    Article  CAS  Google Scholar 

  • Schipper, B., & Schemer, A. K. F. (1966). Effect of antifungal properties of soil on dissemination of pathogen and seedling infection originating from Verticillium-infected achenes of Senecio. Phytopathology, 56, 549–552.

    Google Scholar 

  • Schoch, C. L., Sung, G. H., López-Giráldez, F., Townsend, J. P., & Sugiyama, J. (2009). The Ascomycota tree of life: a phylum-wide phylogeny clarifies the origin and evolution of fundamental reproductive and ecological traits. Systematic biology, syp020.

  • Schreiber, L. R., & Green, R. J. (1963). Effect of root exudates on germination of conidia and microsclerotia of Verticillium albo-atrum inhibited by soil fungistatic principle. Phytopathology, 53, 260–264.

    Google Scholar 

  • Sorensen, L. H., Schneider, A. T., & Davis, J. R. (1991). Influence of sodium polygalacturonate sources and improved recovery of Verticillium species from soil. Phytopathology, 81, 1347.

    Google Scholar 

  • Steventon, L. A., Fahleson, J., Hu, Q., & Dixelius, C. (2002). Identification of the causal agent of Verticillium wilt of winter oilseed rape in Sweden, V. longisporum. Mycological Research, 106, 570–578.

    Article  CAS  Google Scholar 

  • Subbarao, K. V., Chassot, A., Gordon, T. R., Hubbard, J. C., & Bonello, P. (1995). Genetic relationships and cross pathogenicities of Verticillium dahliae isolates from cauliflower and other crops. Phytopathology, 85, 1105–1112.

    Article  Google Scholar 

  • Watanabe, T., Ozawa, M., & Sakai, R. (1973). New disease of Chinese cabbage caused by Verticillium albo-atrum and some factors related to the incidence of the disease. Annal of Phytopathologicial Society of Japan 120–122.

  • Zeise, K., & Von Tiedemann, A. (2002). Host specialization among vegetative compatibility groups of Verticillium dahliae in relation to Verticillium longisporum. Journal of Phytopathology, 150(3), 112–119.

    Article  Google Scholar 

Download references

Acknowledgments

This work was supported in part by grants from the National Science-technology Support Plan Project (No. 2012BAD50G01), the Natural Science Foundation of China (No. 31401867), the National High Technology Research and Development Program of China (863 Program, No. 2012AA100103), and the Fund of China Agriculture Research system(CARS-25-A-11).

Author information

Author notes

    Authors and Affiliations

    1. Beijing Vegetable Research Center (BVRC), Beijing Academy of Agriculture and Forestry Science (BAAFS), Beijing, 100097, China

      Shuancang Yu, Tongbing Su, Juan Chen, Weihong Wang, Fenglan Zhang, Yangjun Yu, Deshuang Zhang, Xiuyun Zhao & Guixiang Lu

    2. Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (North China), Ministry of Agriculture, Beijing, 100097, China

      Shuancang Yu, Tongbing Su, Juan Chen, Weihong Wang, Fenglan Zhang, Yangjun Yu, Deshuang Zhang, Xiuyun Zhao & Guixiang Lu

    Authors
    1. Shuancang Yu
      View author publications

      You can also search for this author in PubMed Google Scholar

    2. Tongbing Su
      View author publications

      You can also search for this author in PubMed Google Scholar

    3. Juan Chen
      View author publications

      You can also search for this author in PubMed Google Scholar

    4. Weihong Wang
      View author publications

      You can also search for this author in PubMed Google Scholar

    5. Fenglan Zhang
      View author publications

      You can also search for this author in PubMed Google Scholar

    6. Yangjun Yu
      View author publications

      You can also search for this author in PubMed Google Scholar

    7. Deshuang Zhang
      View author publications

      You can also search for this author in PubMed Google Scholar

    8. Xiuyun Zhao
      View author publications

      You can also search for this author in PubMed Google Scholar

    9. Guixiang Lu
      View author publications

      You can also search for this author in PubMed Google Scholar

    Corresponding author

    Correspondence to Fenglan Zhang.

    Additional information

    Shuancang Yu and Tongbing Su contributed equally to this work.

    Electronic supplementary material

    Below is the link to the electronic supplementary material.

    Supplementary Figure 1

    Sequence analysis of mt-SSU rDNA and cytochrome b genes. (DOC 384 kb)

    Supplementary Figure 2

    Sensitivity and specificity detection of HW1-F/HW1-R (DOC 265 kb)

    Rights and permissions

    Reprints and permissions

    About this article

    Check for updates. Verify currency and authenticity via CrossMark

    Cite this article

    Yu, S., Su, T., Chen, J. et al. Real-time PCR as a diagnostic tool for evaluating the resistance of Chinese cabbage cultivars to Verticillium wilt. Eur J Plant Pathol 143, 549–557 (2015). https://doi.org/10.1007/s10658-015-0706-8

    Download citation

    • Accepted:

    • Published:

    • Issue Date:

    • DOI: https://doi.org/10.1007/s10658-015-0706-8

    Keywords

    Search

    Navigation